Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine- Containing Antibody Drug Conjugates Mary Jane Masson Hinrichs1, Pauline M

Published OnlineFirst June 19, 2017; DOI: 10.1158/1078-0432.CCR-17-0219

Cancer Therapy: Preclinical Clinical Cancer Research Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine- Containing Antibody Drug Conjugates Mary Jane Masson Hinrichs1, Pauline M. Ryan1, Bo Zheng2, Shameen Aﬁf-Rider1, Xiang Qing Yu2, Michele Gunsior2, Haihong Zhong3, Jay Harper3, Binyam Bezabeh4, Kapil Vashisht1, Marlon Rebelatto1, Molly Reed1, Patricia C. Ryan1, Shannon Breen3, Neki Patel5, Cui Chen3, Luke Masterson5, Arnaud Tiberghien5, Phillip W. Howard5, Nazzareno Dimasi4, and Rakesh Dixit1

Abstract

Objective: To use preclinical models to identify a dosing gated to SG3249 (rats) or SG3400, a structurally related PBD schedule that improves tolerability of highly potent pyrroloben- (monkeys). zodiazepine dimers (PBDs) antibody drug conjugates (ADCs) Results: Observations of similar antitumor activity in mice without compromising antitumor activity. treated with single or fractionated dosing suggests that antitumor Experimental Design: A series of dose-fractionation studies activity of PBD ADCs is more closely related to total exposure were conducted to investigate the pharmacokinetic drivers (AUC) than peak drug concentrations (Cmax). In contrast, im- of safety and efficacy of PBD ADCs in animal models. The proved survival and reduced toxicity in rats and monkeys treated exposure–activity relationship was investigated in mouse with a fractionated dosing schedule suggests that tolerability of xenograft models of human prostate cancer, breast cancer, PBD ADCs is more closely associated with Cmax than AUC. and gastric cancer by comparing antitumor activity after Conclusions: We provide the first evidence that fractionated single and fractionated dosing with tumor-targeting ADCs dosing can improve preclinical tolerability of at least some conjugated to SG3249, a potent PBD dimer. The exposure– PBD ADCs without compromising efficacy. These findings tolerability relationship was similarly investigated in rat and suggest that preclinical exploration of dosing schedule could monkey toxicology studies by comparing tolerability, as be an important clinical strategy to improve the therapeutic assessed by survival, body weight, and organ-specifictoxi- window of highly potent ADCs and should be investigated cities, after single and fractionated dosing with ADCs conju- further. Clin Cancer Res; 1–11. 2017 AACR.

Introduction the success of the two marketed products, clinical development of ADCs continues to be impeded by inability to escalate to Antibody drug conjugates (ADCs) are a rapidly growing class dosagerangeexpectedtoachievedesiredtherapeuticeffectsdue of targeted anticancer therapeutics that now account for a to safety issues (2). Of the greater than 50 ADCs evaluated in significant fraction of pharmaceutical pipelines (1). Much of early clinical trials, approximately 20 have been discontinued the interest in this technology stems from the recent marketing due to lack of efficacy and/or intolerable toxicity (3). Optimiz- approvals of ado-trastuzumab emtansine (Kadcyla) for the ing dosing schedule could be an important strategy to decrease treatment of metastatic breast cancer and brentuximab vedotin adverse events while maintaining exposure and efficacy (4). (Adcetris) for the treatment of Hodgkin's lymphoma. Despite Doing so requires a greater understanding of the relationship between exposure and toxicity in order to inform the clinical dosing schedule. The most common clinical dosing schedule 1Biologics Safety Assessment, MedImmune, Gaithersburg, Maryland. 2Clinical for ADCs is every 3 weeks (3). This schedule results in high peak 3 Pharmacology and DMPK, MedImmune, Gaithersburg, Maryland. Oncology plasma concentrations with minimal-to-no accumulation with Research, MedImmune, Gaithersburg, Maryland. 4Antibody Discovery and Pro- repeated dosing (5). Although every 3 weeks dosing is consis- tein Engineering, MedImmune, Gaithersburg, Maryland. 5Spirogen Ltd., QMB Innovation Centre, London, United Kingdom. tent with dosing schedules used for most chemotherapy regimens, it is unclear whether this represents the ideal dosing Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). regimen for ADCs. In addition, there is very limited clinical data available to examine the potential benefits of alternative M.J.M. Hinrichs and P.M. Ryan contributed equally to this article. dosing schedules (3, 6, 7). SAR3419, an anti-CD19 ADC conju- Corresponding Author: Mary Jane Masson Hinrichs, MedImmune LLC, 1 Med- gated to the maytansinoid DM4, is an example where use of Immune Way, Gaithersburg, MD 20878. Phone: 2022957576; Fax: 3013981111; alternative dosing schedule positively impacted clinical devel- E-mail: [email protected] opment of an ADC. In the first in human trial, the maximum doi: 10.1158/1078-0432.CCR-17-0219 tolerated dose (MTD) was 160 mg/m2 on a every 3 weeks 2017 American Association for Cancer Research. dosing schedule (8). Although the overall response rate (ORR)

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risk of safety issues due to steep dose–response curves. Therefore, Translational Relevance understanding the relationship between exposure and tolerability Optimization of dosing schedule could be an important is essential to selecting a rational dosing schedule. We conducted a mitigation strategy to improve tolerability of ADCs with series of preclinical safety and efficacy studies to better understand narrow therapeutic windows, especially those conjugated to the relationship between exposure and therapeutic index of PBD highly potent payloads such as pyrrolobenzodiazepine dimers ADCs. Our aim was to identify whether dosing schedule can (PBDs). Doing so requires a greater understanding of the improve tolerability of highly potent ADC without compromising exposure–response relationship to enable selection of a dosing antitumor activity. regimen that decreases adverse events while maintaining efficacy. Assessing this relationship in patients can be lengthy and resource intensive; therefore, we evaluated whether preclinical Materials and Methods models could be used to understand the pharmacokinetic Test articles drivers of safety and efficacy for PBD ADCs in animals. By The anti-EphA2 (1C1), anti-5T4 (5T4), and isotype control conducting a series of dose-fractionation studies, we deter- (R347) antibodies used in these studies have been previously mined that fractionated dosing improved tolerability of PBD described (16–18). 1C1 cross-reacts with rat, monkey, and ADCs without impacting antitumor activity. Together, these human EphA2; 5T4 is only cross-reactive with monkey and results suggest that fractionated dosing could widen the ther- human 5T4 (16, 19). These antibodies were engineered to apeutic window of PBD ADCs. enable site-specific conjugation of two PBD dimers per antibody. The maleimide-PEG8 PBD dimers used for conjugation were SG3249, also known as tesirine (20), and SG3400, a PBD dimer based on SG2000 (21, 22). Site-specific conjugation was carried out as described previously (17). Briefly, antibodies were at this dose level was 22%, findings of grade 2/3 ocular toxicity reduced using 40 molar equivalents of Tris (2-carboxy-ethyl)- prevented escalation to higher, potentially more efficacious phosphine (TCEP) in PBS pH 7.2, 1 mmol/L ethylenediamine dose levels (9). In an attempt to improve tolerability, a frac- tetraaceticacid (EDTA) for 3 hours at 37 C. Following overnight tionated dose schedule was evaluated in which patients were dialysis in PBS pH 7.2, 1 mmol/L EDTA at 4 C using 10,000 administered four weekly doses of 55 mg/m2 followed by four MWCO dialysis cassettes, 20 molar equivalents of dehydroas- additional doses every 2 weeks. Use of this schedule appeared corbic acid were added for 4 hours at 25 C. The solution was to have a positive impact as the ORR increased to 33%, and the filtered through a 0.2-mmsyringefilter and eight equivalents of severity of ocular toxicity decreased to mostly grade 1 events. SG3249 or SG3400 were sequentially added, followed by incu- Despite the success with SAR3419, reports with other ADCs bation at room temperature for 1 hour under gentle rotation. have been mixed. In the case of gemtuzumab ozogamicin The conjugation was quenched by the addition of 4 molar (Mylotarg), an anti-CD33 antibody conjugated to calicheami- equivalents (over SG3249 or SG3400) of N-acetyl cysteine. Free cin, early data from a multicenter phase II uncontrolled trial in unreacted SG3249 and SG3400 and macromolecular aggregates 57 adult patients with acute myeloblastic leukemia (AML), were removed using ceramic hydroxyapatite type II chromatog- patients demonstrated that fractionated dosing with mono- raphy as described previously (18). Site-specific ADCs were therapy Mylotarg significantly improved safety without impact- formulated at 3 mg/mL in PBS pH 7.2. The ADCs were char- ing efficacy (10). However, follow-up data from a small retro- acterized using complementary analytical methods and analyt- spective study in relapsed/refractory patients with AML treated ical data are shown in Supplementary Figure S1. with Mylotarg in combination with chemotherapy, using either the standard every 3 weeks (18 patients) or a fractionated (15 Animals patients) dosing schedule, failed to demonstrate significant All studies were conducted at facilities that comply with the differences in outcomes or safety between the two schedules principles of the "Guide for Care and Use of Laboratory Animals" (11, 12). Although these data demonstrate that alternative and are accredited by the Association for Assessment and Accred- dosing regimens do not always improve the therapeutic index itation of Laboratory Animal Care International (AAALAC). All of ADCs, it is clear that further exploration of the relationship is study protocols were approved by the testing facilities Institu- warranted given the high attrition rate of clinical stage ADCs. tional Animal Care and Use Committee. For xenograft efficacy Recently, there has been significant interest in developing next- studies, 5- to 6-week-old female athymic (nu/nu) mice were generation ADCs with increased potency and alternative mechan- obtained from Harlan Sprague–Dawley Inc. For patient-derived isms of action. Pyrrolobenzodiazepine dimers (PBDs) are a class xenograft (PDX) efficacy studies, 6- to 8-week-old female BALB/c of DNA crosslinking warheads that are significantly more potent nude mice were obtained from Shanghai Sino-British SIPPR/BK than the tubulin-inhibiting payloads used in most clinical-phase Laboratory Animal Co., Ltd. For safety studies, 8- to 12-week-old ADCs (13). Interest in PBD dimers has grown significantly over male Sprague–Dawley rats were obtained from Harlan Labora- the past year following the release of promising clinical data from tories and 2- to 4-year-old male cynomolgus monkeys (Macaca trials with PBD conjugates, rovalpituzumab tesirine (14) and fascicularis) of Cambodian origin were obtained from a commer- vadastuximab talirine (SGN-CD33A; ref. 15), in difficult-to-treat cial supplier. cancers, such as small cell lung cancer and relapsed, refractory acute myeloid leukemia, respectively. As a result, it is anticipated Mouse efficacy studies þ that a significant number of PBD ADCs will advance into clinical EphA2 prostate cancer xenograft model: 5 106 PC3 (human development over the next few years. Although increased potency prostate cancer cell lines) were implanted subcutaneously into the þ can enhance the antitumor activity of an ADC, it also increases the right flank of mice (seven per group). 5T4 breast cancer xenograft

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model: mice were supplemented with 60-day 0.36-mg slow- effects were assessed by comparing safety endpoints to release estradiol pellets the day before 1 107 MDA-MB-361 prestudy values. All animals were evaluated for clinical signs, tumor cells in 50% Matrigel were implanted subcutaneously into changes in body weight, clinical pathology, food consumption, þ the second mammary fat pad (10 per group). 5T4 gastric cancer ophthalmology assessments, pharmacokinetic analysis, gross xenograft model: 5 106 N87 cells in 50% Matrigel were injected pathology with organ weights, and microscopic observations. subcutaneously into the second mammary fat pad of mice (five Blood samples were collected for clinical pathology via a per group). Target expression was confirmed by immunohis- single draw and divided into K2 EDTA tubes for hematology, þ þ tochemistry as previously described (16, 19). Low 5T4 (1 by serum separator tubes for serum chemistry, and sodium citrate IHC) ST-02-009 gastric PDX model: mice (10 per group) were tubes for coagulation. Hematology and serum chemistry samples implanted subcutaneously into right flank with ST-02-009 pas- were collected and analyzed weekly beginning on day 8 to day 72. 3 þ þ sage 4 (P4) tumor slices (30 mm ). High 5T4 (3 by IHC) Blood samples for pharmacokinetic analysis were collected in K2 ST-02-0164 gastric PDX model: mice (10 per group) were EDTA tubes at multiple time points on days 1, 2, 3, 8, 15, 16, 22, implanted subcutaneously into right flank with ST-02-0164 P4 and 36. tumor slices (30 mm3). When the mean tumor volume was A gross necropsy was performed on day 72 on all main study about 150 to 200 mm3, tumor-bearing mice were randomized animals and organs, including brain, lung, liver, kidney, spleen, into treatment groups. Test articles were administrated intrave- thymus, testes, heart, and bone, were embedded in paraffin, nously at indicated dose and dosing frequency (SD, single dose; sectioned, stained with hematoxylin and eosin, and examined or FD, fractionated weekly doses 3). Dose levels for each ADC by light microscopy. were chosen based on minimum efficacious dose (MED) determined in single-dose pilot studies. Tumors were measured twice Statistical analysis weekly using calipers. Tumor volumes were calculated using the Data from safety and efficacy studies are reported as means formula 0.5 x L x W2 (L is the length; W is the width). Animals SEM. Statistical analyses comparing means between three or more were euthanized when tumor volumes reached approximately groups were performed using one-way ANOVA with Newman– 2,000 mm3. Keuls post hoc test. All analyses were performed with Prism 6 software (GraphPad Software). Differences were considered sig- Rat safety study nificant when #, P < 0.05. Male Sprague–Dawley rats (six per group) were administered either a single intravenous injection (day 1) of 1.5 mg/kg or three Quantitation of 1C1-SG3249 ADC, total antibody, and weekly intravenous injections (days 1, 8, and 15) of 0.5 mg/kg warhead concentrations in rat plasma anti-EphA2 antibody conjugated to SG3249 (1C1-SG3249). Tox- 1C1-SG3249 ADC, total antibody, and free warhead icokinetic (TK) satellite animals (six per group; PK sparse sam- (SG3199) in rat K2EDTA plasma samples were quantitated using pling/three samples per timepoint) were included in each treat- LC/MS-MS technology as described previously (17). Briefly, ment arm to measure plasma concentration of total antibody and samples were diluted with 0.1% BSA/Tris-Tween buffer and ADC. Control rats (six per group) were administered a single split into two aliquots. Total antibody quantification was per- intravenous injection of vehicle control on day 1. All main study formed in SILuMab (Sigma Aldrich) internal standard-spiked animals were evaluated for clinical signs, changes in body weight, samples. Following magnetic Protein A bead capture, a trypsin clinical pathology, gross pathology with organ weights, and digest was performed to generate characteristic peptides microscopic observations. All TK satellite animals were evaluated (VVSVLTVLHQDWLNGK and TTPPVLDSDGSFFLYSK) used as for clinical signs, changes in body weight, and pharmacokinetic quantitative surrogates of total antibody concentration. For analysis. quantitation of ADC, samples were immunoprecipitated with Hematology and serum chemistry samples were collected and Protein A magnetic beads followed by papain digestion to analyzed on days 8 and 15. These timepoints were chosen to release warhead. The concentration of the released drug is evaluate the effects of repeated dosing with 1C1-SG3249 without proportional to the antibody conjugated drug concentration. exceeding blood draw limits for rats. Blood samples for pharma- Deuterium-labeled warhead was used as an internal standard for cokinetic analysis were collected from TK satellite animals in K2 ADC quantitation. The final extracts for analytes were quanti- EDTA tubes at multiple time points on days 1, 2, 3, 8, 15, 16, 22, tated using HPLC with triple quadrupole MS/MS detection using and 29. positive ion electrospray. A linear, 1/concentration2 weighted A gross necropsy was performed on day 29 on all main study regression was used to quantitate unknown samples from the animals and organs, including brain, lung, liver, kidney, spleen, standard curve. The calibration range was 0.05 to 10 mg/mL for thymus, testes, heart, and bone, were embedded in paraffin, total antibody and 0.051 to 10.2 mg/mL for ADC. Dilutional sectioned, stained with hematoxylin and eosin, and examined linearity was verified with ultra-high QC samples during assay by light microscopy. qualification; any sample with a concentration above the upper limit of the calibration range was diluted 10-fold to obtain Monkey safety study results within the established assay limits. Final results, which Male cynomolgus monkeys (three per group) were admin- are reported in neat matrix, are inclusive of the applied dilution. istered either a single intravenous injection of 4.5 mg/kg (day Free warhead was measured by LC/MS-MS as described previ- 1) or three weekly intravenous injections of 1.5 mg/kg (days 1, ously (17) using deuterium-labeled SG3199 as an internal 8, and 15) R347 antibody conjugated to SG3400 (R347- standard. Free warhead/internal standard peak area ratios were SG3400) via the saphenous vein. Because of ethical considera- utilized for the construction of calibration curves, using weight- tions associated with nonhuman primate (NHP) use, no con- ed (1/X2) linear least-squares regression. The lower limit of trol animals were assigned to the study and treatment-related quantitation (LLOQ) of free warhead is 0.2 nmol/L.

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Quantitation of R347-SG3400 total antibody concentrations in 1.5 mg/kg or as three weekly fractionated doses of 0.5 mg/kg cynomolgus monkey plasma (Fig. 1A). Although both dosing schedules were equally effec- Total antibody concentrations of R347-SG3400 in cynomol- tive over the long term, the time-to-tumor regression was gus monkey K2EDTA plasma were determined using a qualified slightly slower in mice treated with three weekly doses com- universal ELISA method. An ELISA method was chosen for this pared to mice treated with an SD of 1C1-SG3249 (day 43 vs. day study after comparability studies demonstrated no significant 32, respectively). As expected, limited antitumor activity was differences between ELISA and LC/MS methods. Briefly, sheep observed in mice treated with negative control R347-SG3249. anti-human IgG (HþL) antibody (The Binding Site) was used In the human gastric and breast cancer xenograft models, to coat Nunc Maxisorp plates up to 72 hours at 2 to 8Cata mice were treated with an anti-5T4 PBD ADC (5T4-SG3249) concentration of 1 mg/mL in 50 mmol/L carbonate buffer. as an SD of 1 mg/kg or as three weekly fractionated doses of Following a block step with I-Block buffer, samples (diluted 0.33 mg/kg (Fig. 1B and C). As with the PC3 prostate model, 1/10 in I-Block) were incubated on the blocked plate. The single and fractionated dosing schedules were equally effective captured R347-SG3400 antibody was detected with goat over the study period, although the time-to-tumor regression anti-human IgG (HþL) horseradish peroxidase (HRP, Bethyl was again slightly faster after single-dose treatment in both Laboratories). Tetramethylbenzidine chromogenic substrate models. The impact of dosing regimen on antitumor activity was used to measure the binding complex; once stopped with was further evaluated by comparing single and fractionated 2NH2SO4, the plate was read at 450 and 650 nm, and the dosing schedules of 5T4-SG3249 in two gastric PDX models intensity of color development was directly proportional to with variable 5T4 expression (Fig. 1D and E). As in xenograft levels of total antibody in sample. Dilutional linearity was models, fractionated dosing resulted in comparable anti- verified with ultra-high QC samples during assay qualification. tumor activity with longer time-to-tumor regression in the low Sample results were reported in ng/mL; the lower limit of 5T4-expressing tumor model (Fig. 1D). In contrast, no differ- quantitation was 50 ng/mL. Total ADC and free warhead were ences in antitumor activity were observed in the high 5T4- not assessed in this study as previous in vivo studies confirmed expressing tumor after single or fractionated dosing (Fig. 1E). that our site specific PBDs are highly stable with limited de- Moreover, tumor regrowth was significantly delayed in mice conjugation (17). As a result, total antibody can be used as a treated with fractionated dosing. Although there is a trend reliable surrogate for ADC exposure. toward slightly delayed onset of antitumor activity after fractionated dosing, overall, the data indicate that single and fractionated Pharmacokinetic analysis dosing schedules of targeted PBD ADCs have comparable anti- A standard noncompartmental analysis (NCA) for a bolus tumor activity in human cancer mouse models. intravenous administration was used for all pharmacokinetic data analysis using Phoenix 64 WinNonlin 6.3 (Pharsight). The Fractionated dosing improved the tolerability of 1C1-SG3249 maximum observed peak plasma concentration (Cmax), area in rats under the plasma concentration versus time curve (AUC) Next, we evaluated whether fractionated dosing affected the – and half-life (t1/2) of elimination phase were estimated and tolerability of 1C1-SG3249 in a rat toxicity study (Fig. 2A F). — summarized statistically as mean and 1 SD. Rat AUC0-28 was For this, we compared body weight gain aprimarytoxicology — calculated using NCA and compared to AUC0-28 calculated endpoint used to assess overall health of animals (23) across using population PK analysis to verify NCA did not overesti- three groups of rats (six per group) treated with vehicle control, mate the exposure (AUC) in rats. Because the two values were an SD of 1.5 mg/kg 1C1-SG3249 or three weekly fractionated similar, only NCA-estimated rat AUC0-28 is reported in the doses of 0.5 mg/kg 1C1-SG3249. Treatment with an SD of results. Because of limited monkey PK time points, monkey 1.5 mg/kg SG3249 had the greatest impact on body weight gain PK data was modeled using a two-compartment population relative to vehicle control rats (Fig. 2B). Animals administered PK model with linear elimination (NONMEM 7.2) and model- an SD of 1.5 mg/kg experienced a significant reduction based population predictions (PRED) were used to monkey (10%; P < 0.05 vs. control) in body weight gain starting on calculate AUC0-28. day 8. These effects became increasing severe over time (up to 20% on day 11; P < 0.001vs.control)andpersisteduntilthe end of study. A significant reduction in body weight gain was Results also detected in rats treated with fractionated dosing; however, Tumor regression in mice is comparable following single or the effects were generally milder and delayed in onset com- fractionated dose regimens of tumor-targeting PBD ADCs pared to single-dose treatment. From day 11 onward, a small Mouse xenograft and PDX models of human cancer were used decrease in body weight gain (3%; P < 0.05 vs. control) was to compare the antitumor activity of PBD ADCs after single and observed that continued to decline (up to 10% P < 0.001 vs. fractionated dosing (Fig. 1). Preliminary studies were con- control) until the end of study. Myelotoxicity was the primary þ ducted in cell-line-derived xenograft models of EphA2 human dose-limiting toxicity observed in rats (Fig. 2C–F). Hemato- þ prostatecancer(Fig.1A),aswellas5T4 human gastric (Fig. 1B) logic analysis on days 8 and 15 demonstrated that treatment and breast (Fig. 1C) cancer, to evaluate the exposure-activity with an SD of 1.5 mg/kg 1C1-SG3249 resulted in significant across target antigens and tumor types. Results were similar decreases in several hematologic parameters, including hemat- across models, with both dosing schedules resulting in signif- ocrit, neutrophils, white blood cells, and reticulocytes. The 3 icant antitumor activity that persisted until day 61 of study. severe neutropenia (<0.5 cells * 10 /mL) observed on days 8 In the human prostate cancer model, mice were treated with and 15 was likely a significant factor contributing to early anti-EphA2 PBD ADC (1C1-SG3249) or negative control R347 mortality secondary to infection in these animals (Fig. 2C). In PBD ADC (R347-SG3249) administered as either an SD of contrast, fractionated dosing was associated with less severe

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ABVehicle control Vehicle control CVehicle control 1C1-SG3249 SD (1.5 mg/kg) 5T4-SG3249 SD (1 mg/kg) 5T4-SG3249 SD (1 mg/kg) 1C1-SG3249 FD (0.5 mg/kg x 3) 5T4-SG3249 FD (0.33 mg/kg x 3) 5T4-SG3249 FD (0.33 mg/kg x 3) R347-SG3249 SD (1.5 mg/kg) 1,800 R347-SG3249 FD (0.5 mg/kg x 3) 1,200 1,500 ) ) ) 3 3 1,500 3 1,200 900 1,200 900 900 600 600 600 300

Tumor volume (mm 300 Tumor volume (mm 300 Tumor volume (mm

0 0 0 22 29 36 43 50 57 64 14 21 28 35 42 49 56 63 Days after inoculation Days after inoculation Days after inoculation

Vehicle control E Vehicle control D 5T4-SG3249 SD (1 mg/kg) 5T4-SG3249 SD (1 mg/kg) 5T4-SG3249 FD (0.33 mg/kg x 3) 5T4-SG3249 FD (0.33 mg/kg x 3) R347-SG3249 SD (1 mg/kg) R347-SG3249 SD (1 mg/kg) R347-SG3249 FD (0.33 mg/kg x 3) R347-SG3249 FD (0.33 mg/kg x 3) 2,400 2,000 ) ) 3

3 2,000 1,500 1,600

1,200 1,000

800 500 Tumor volume (mm Tumor volume (mm 400

0 0 24 31 38 45 52 59 66 73 80 36 43 50 57 64 71 78 85 Days after inoculation Days after inoculation

Figure 1. Comparable antitumor activity after single or fractionated dosing with PBD ADCs in preclinical mouse tumor models. A–E, Single (SD) and fractionated (FD) dosing schedules were evaluated in (A) an EphA2þ human prostate cancer cell line (PC3) xenograft model, (B)a5T4þ human gastric cancer N87 xenograft model, (C)a5T4þ human breast cancer MDA-MB-361 xenograft model, and (D, E) two PDX gastric cancer models with (D)low5T4 expression or (E) high, heterogeneous 5T4 expression, as determined by immunohistochemistry. When the mean tumor volume reached 150 to 200 mm2, female athymic (nu/nu) mice (5–10/group) were treated with an SD or FD doses of vehicle control or a tumor-targeting PBD ADC [(A)1C1-SG3249, (B–E) 5T4-SG3249] by intravenous injection. Where indicated (A, D, E), animals were similarly treated with an isotype-matched PBD ADC (R347-SG3249) to serve as a negative control group. Results are represented as the mean SEM of each group at individual timepoints. myelosuppression as noted by less impact on the same hema- mild to moderate macroscopic observations of bilateral corneal tologic parameters. In the case of neutrophils, although there opacity corresponding with acute inflammation of the anterior was some decrease compared to controls, mean absolute cells segment (Supplementary Table S1). This finding was unexpect- counts remained within normal range for male rats (0.84–2.67 ed as ocular toxicity has not been reported as an adverse event 3 cells * 10 /mL; ref. 24) at both timepoints. In addition, no signs of PBD ADCs in animals and was not observed in rats treated of cumulative bone marrow toxicity were observed after repeat- with isotype control R347-SG3249 in a pilot single-dose tox- ed dosing on day 15 compared to day 8. Histopathologic icity study (Supplementary Table S1). It is possible that this analysis of bone marrow was performed; however, a direct finding could be related to normal tissue expression as EphA2 comparison of fractionated and single-dose regimens was not expression has been reported in corneal epithelial cells (25) possible as all animals were necropsied on day 29, after and loss of EphA2 receptor function has been associated with extensive recovery had occurred. cataract development (26). Although it is unclear why fraction- Although fractionated dosing improved overall survival and ated dosing improved some organ-specific toxicities and not myelotoxicity in rats, it did not appear to impact ocular toxicity others, it is possible that dosing schedule might not have as associated with 1C1-SG3249 treatment. Histopathology anal- great an influence on target-dependent toxicities due to recep- ysis indicated that both dosing regimens were associated with tor-mediated uptake (27).

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Figure 2. Fractionated dosing improves tolerability of PBD ADC in rats. Rats (n ¼ 6) were treated with a single intravenous injection (1.5 mg/kg SD) or three weekly fractionated intravenous injections (0.5 mg/kg FD) of an anti-EphA2 PBD ADC (1C1- SG3249). Control animals were treated with a single intravenous injection of vehicle control. A and B, Tolerability was assessed by measuring percent (A)survivaland(B)bodyweightgainin rats over a 29-day period. A total of four animals treated with an SD of 1.5 mg/kg 1C1-SG3249 were euthanized due to moribund condition or found dead between days 18 and 29 of study. Data points represent the mean value of each group at individual timepoints; error bars, mean SEM. C–F, Improved tolerability of fractionated dosing associated with reduced impact on peripheral blood cell counts. Hematologic analysis was conducted on peripheral blood samples collected on days 8 and 15. Bars represent the mean value of each group; error bars, mean SEM. #, P < 0.05 versus SD-treated rats.

Fractionated dosing also improves tolerability of a structurally R347, an IgG1 isotype control antibody (29), was chosen for related PBD ADC in monkeys the antibody component in order to eliminate potential con- A follow up toxicology study was conducted in cynomolgus founding factors related to target expression. This construct was monkeys to assess applicability of these findings across species. then used to test whether fractionated dosing improves toler- We chose monkeys for this study as they are the most com- ability of a structurally related PBD ADC. Male monkeys (three monly used toxicology species to evaluate the safety of bio- per group) were treated with an SD of 4.5 mg/kg or three weekly pharmaceutical products (28); however, due to ethical con- fractionated doses of 1.5 mg/kg of R347 antibody conjugated to siderations associated with use of NHP, we were limited in the SG3400 (R347-SG3400) by intravenous injection. No control number of ADC constructs we could evaluate. Therefore, the animals were included in the study to further minimize use of studywasdesignedtoevaluatewhetherthefindings observed NHP; therefore, treatment-related findings were assessed by with SG3249-based ADC could be applied to other PBD ADCs comparing safety endpoints to prestudy values. while using the minimum number of animals. SG3400 was All monkeys survived to the end of study after single or chosen as the payload as it is structurally similar to SG3249; fractionating dosing with R347-SG3400. However, monkeys trea- however, the in vitro potency of the warhead component ted with an SD exhibited a notable decline in body weight on day (SG2000; ref. 22) is approximately 10-fold less potent than 8 that peaked on day 36 and remained below prestudy values the warhead component of SG3249 (SG3199; refs. 20, 21). throughout the study period (Fig. 3A). In comparison, despite

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Figure 3. Similar improvements in tolerability after fractionated dosing of a structurally related PBD ADC in monkeys. Male monkeys (n ¼ 3) were treated with a single IV injection (4.5 mg/kg SD) or three weekly fractionated intravenous injections (1.5 mg/kg FD) of a nontargeted PBD ADC (R347-SG3400). No control animals were included due to ethical considerations related to use of nonhuman primates. As a result, treatment-related findings were assessed by comparing all values to baseline. A, Tolerability was assessed by measuring percent (A) body weight gain in monkeys over a 72-day period. Data points represent the mean value of each group at individual timepoints; error bars, mean SEM. B–D, Improved tolerability of fractionated dosing associated with decreased kidney injury as assessed by (B) decreased serum creatinine, a biomarker of renal injury, (C) decreased histopathologic observations of tubular degeneration, interstitial fibrosis, and mononuclear infiltration, and (D) reduced monocytosis. Bars represent the mean value of each group; error bars, mean SEM. Histopathology severity classified as 1 ¼ slight, 2 ¼ mild, 3 ¼ moderate, 4 ¼ severity.

an early transient drop in body weight, monkeys treated with a treated with fractionated dosing were significantly lower and did fractionated dosing schedule exhibited marked body weight gain not exceed 1 103/mL at any timepoint. over the course of the study. Clinical signs of discolored skin (black, red), alopecia, squinting of the eyes, and nasal discharge Comparable total exposure after single or fractionated dosing were observed in the animals that received an SD of 4.5 mg/kg of with PBD ADCs in rats and monkeys R347-SG3400. Although similar clinical signs were observed in Plasma concentrations of intact ADC, total antibody (conju- the fractionated dosing group, the findings were less severe and/or gated and unconjugated 1C1 antibody), and free SG3199 war- limited in duration. Together, these results suggest fractionated head were measured in rats (Fig. 4). Following intravenous dosing also improved tolerability of a structurally related, but administration of an SD of 1.5 mg/kg or 3 weekly fractionated chemically distinct, PBD ADC in monkeys. doses of 0.5 mg/kg, 1C1 ADC exhibited linear PK. The area under Fractionated dosing also appeared to improve kidney injury concentration–time curve from time 0-to-7 days post-dose associated with R347-SG3400 treatment (Fig. 3B and C). Mon- (AUC0-7) increased dose proportionally from 30.3 mg day/mL keys treated with an SD of 4.5 mg/kg exhibited a delayed increase at 0.5 mg/kg to 85.2 mgday/mL at 1.5 mg/kg (Supplementary in serum creatinine, a biomarker of kidney injury, on day 22 Table S2). Furthermore, total plasma exposure over 28-day study (FIG. 3B). Serum creatinine levels continued to increase with period (AUC0-28) was comparable following the SD of 1.5 mg/kg time, peaking on day 50 (up to 3 mg/dL), and remaining elevated (125.3 mgday) and 3 weekly fractionated doses of 0.5 mg/kg until the end of the study. In contrast, serum creatinine in mon- (129.9 mgday/mL). As expected, peak plasma concentration keys treated with fractionated dosing remained near baseline until (36.8 mg/mL) after an SD of 1.5 mg/kg was approximately day 72. Histopathologic analysis confirmed that elevations in three-fold higher than that (11.5 mg/mL) of the fractionated dose serum creatinine were associated with kidney injury characterized group. The high Cmax in the single-dose group appeared to by moderate interstitial fibrosis with mononuclear cell infiltra- inversely correspond to the low tolerability, suggesting a Cmax tion, moderate tubular atrophy, and moderate tubular degener- associated tolerability in rats. Free warhead levels remained below ation/regeneration in all animals treated with an SD of R347- the limit of detection (0.2 nmol/L) at all timepoints. This, in SG3400 (Fig. 3C). By comparison, the severity of these findings addition to the comparable pharmacokinetic profile of intact was greatly reduced to mild, or not present, in monkeys treated ADC and total antibody, indicated limited deconjugation of with a fractionated dosing schedule. Treatment with an SD was SG3249 from the ADC. 3 also associated with significant monocytosis (>1 10 cells/mL; Similarly, plasma exposure (AUC0-7) of total antibody following ref. 30), likely secondary to kidney injury, on day 15 that persisted intravenous administration of R347-SG3400 ADC in cynomolgus until day 64 (Fig. 3D). In contrast, monocytes counts in monkeys monkeys increased approximately dose-proportionally from

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meters driving efficacy and toxicity in preclinical models. We used dose-fractionation studies to evaluate the impact of lowering peak drug concentrations while maintaining equivalent total exposure. By doing so, we demonstrated that fractionated dosing improved tolerability of highly potent PBD ADCs while maintaining antitumor activity in a preclinical setting. Because clinical utility is a balance of safety and efficacy, the schedule-dependence of both parameters was evaluated in preclinical models. Although we did not conduct pharmacokinetic analysis in our mouse studies, we anticipate that total exposure or AUC would be similar in mice treated with single or fractionated dosing as ADC PK in tumor-bearing mice is linear and similar to that in nontumor-bearing mice (17, 31). Therefore, on the efficacy side, preliminary data from mouse xenograft and PDX models suggests that antitumor activity of at least some PBD ADC is more closely correlated to total exposure than Cmax (Figs. 1 and 4). However, given that this is based on data from a small number of models, it is unknown how broadly these findings will translate Figure 4. across tumor targets and histologies. To our knowledge, there Comparable exposure in rats treated with single versus fractionated dosing have been no publications exploring the impact of dosing sched- regimens of PBD ADC. Plots of mean concentration vs time of total of ule on preclinical efficacy with PBD ADCs, indicating that addi- total antibody (solid line) and ADCs (dashed line) in rats (six per group) tional work is needed to understand the broad applicability of treated with an SD of 1.5 mg/kg or three weekly (FD) of 0.5 mg/kg these results. However, given the low tumor penetration and 1C1-SG3249 by intravenous injection. Each time point is the mean values limited tissue distribution of antibodies (32), high peak plasma SD of three sparse samples. The dashed line represents the LLOQ, which concentrations are not anticipated to increase antitumor activity was 0.05 mg/mL for both total antibody and ADC. of an ADC (33). Moreover, similar findings have been observed with other classes of ADCs, such as those conjugated to micro- tubule-inhibiting payloads. Data from a recent publication by 80.7 mgday/mL at 1.5 mg/kg to 261.4 mgday/mL at 4.5 mg/kg, Govindan and colleagues (34), demonstrated that antitumor indicating linear PK in monkeys (Table 1). Peak plasma concentra- activity of T-DM1, a HER2-targeting ADC conjugated to the tion of total antibody was about threefold higher or dose- maytansinoid DM1, was similar in animals treated with single proportionally in the single high dose of 4.5 mg/kg group or fractionated dosing schedules. In addition, dose fractionation (102.0 mg/mL) than in the low dose of 1.5 mg/kg (31.1 mg/mL), studies with IMMU-130, a CEACAM5-targeting ADC conjugated which is consistent with reduced tolerability in this group. A to the DNA topisomerase I inhibitor, 7-ethyl-10-hydroxycamp- population PK model was used to fit observed PK data, and tothecin (SN-38), have also shown comparable antitumor activity AUC0-28 was thus calculated based on population prediction with single and fractionated dosing regimens (35). Taken togeth- (PRED) of the population PK model (Supplementary Fig. S2 er, these data suggest preclinical anti-tumor activity of ADCs could and Table 1). Using this model, it is predicted that AUC0-28 be more closely associated with total exposure rather than the should be comparable after single (581.9 mgday/mL) or frac- peak concentration. tionated dosing (526 mgday/mL). Collectively, PK and safety On the safety side, preliminary data from rat and monkey fi data across species of rat and monkey implicate Cmax-driven toxicology studies suggest that dosing schedule can also signi - tolerability for PBD ADCs. cantly impact preclinical tolerability of PBD ADCs. Fractionated dosing was associated with a significant improvement in body weight changes in both species. Pharmacokinetic analysis in rats Discussion and monkeys revealed that Cmax, but not AUC, appeared to Despite the highly targeted nature of ADCs, clinical develop- correlate with preclinical tolerability, suggesting that tolerability ment of these molecules continues to be limited by narrow of ADCs may be more closely associated with Cmax then total therapeutic indexes (2, 3). Having a better understanding of the exposure. Organ-specific toxicities were also affected by dosing relationship between exposure and toxicity could play a vital role schedule. In rats, where bone marrow suppression is a major dose- in early clinical trial design by enabling rational selection of limiting toxicity of PBD ADCs (36), fractionated dosing showed dosing schedules. Our studies investigated whether preclinical significantly less effects of 1C1-SG3249 on peripheral blood studies could be used to understand the pharmacokinetic para- counts compared to the single-dose group. In monkeys,

Table 1. Pharmacokinetics of R347-SG3400 after single or fractionated dosing in monkeys (three/group) a Treatment Analyte Cmax (mg/mL) t1/2 (day) AUC0–7 (day mg/mL) AUC0–28 (day mg/mL) 4.5 mg/kg SD Total Ab 102.0 (11.2) 10.4 (0.8) 261.4 (37.6) 581.9b 1.5 mg/kg FD Total Ab 31.1 (4.2) 80.7 (8.6) 526.1b PK parameters presented as mean (SD). a Cmax after ﬁrst dose. b AUC0-28 calculation was based on population predictions (PRED) of PK model.

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fractionated dosing with a structurally related ADC also improved that make it complicated to extract a single simple pharmacoki- organ-specific toxicities—thus confirming that this finding is not netic parameter directly from clinical safety findings (45). restricted to a particular PBD or to a single species. For this study, Another issue that remains to be addressed is whether dose we used a nontargeted PBD ADC (R347-SG3400) to eliminate the fractionation can improve both on and off target toxicities. Given potential for confounding data related to target expression on the limited number of constructs that we could analyze in this normal tissues. Unlike SG3249, the major toxicity of R347- study we cannot rule out that dose fractionation might not SG3400 in monkeys was renal injury. This difference, although improve all toxicities, in particular on-target toxicities. While the not predictable, was not entirely unexpected as the safety profile of effect of target expression on exposure–toxicity relationships cytotoxic agents can be species-specific and/or related to mod- should be further explored, it is likely a very complex issue given ifications in chemical structure (37). The ability of fractionated on-target toxicity is not driven solely by target expression and that dosing to reduce kidney toxicity of an SG3400 ADC was remark- other factors (e.g., proliferative/regenerative potential of target able. A SD of R347-SG3400 resulted in delayed kidney injury that cell/organ, mechanism/potency of warhead, and accessibility of was completely absent in animals treated with a fractionated the ADC to the target cell) could play a role (46). Therefore, at dosing regimen. This suggests that peak plasma concentration is present, the impact of dose-fractionation on the preclinical TI of a major factor driving tubular degeneration in the kidneys of PBD ADCs should be empirically tested for novel ADCs with animals treated with PBD ADCs. Together, these data indicate that different patterns of normal tissue target expression. fractionated dosing can have an overall positive effect on both In conclusion, these data indicate preclinical dose-fraction- tolerability and organ-specific toxicities across species with dif- ation studies could be an important strategy to inform selection ferent PBD ADCs. of clinical dosing schedule by increasing our understanding of the In addition, the translatability of these findings to patients is underlying pharmacokinetic drivers of safety and efficacy for PBD not known given the uncertain predictive value of animal models ADCs. While additional work is needed to understand the broad (38). Xenograft mouse models, in particular, are generally not applicability of these data, our work suggests that fractionated considered reliable predictors of clinical activity due to the sig- dosing could be an important mitigation strategy to widen the nificant differences between mouse and human tumor growth therapeutic index of highly potent ADCs in patients. characteristics and metastatic behavior. Translatability of preclinical activity of conjugated antibodies is further complicated by fl fi Disclosure of Potential Con icts of Interest signi cant differences in target distribution and pharmacokinetic fl fi fi A. Tiberghien has ownership interest in AstraZeneca. No potential con icts of pro les across species (39). Despite these uncertainties, con - interest were disclosed by the other authors. dence in preclinical outcomes can be greatly increased by using a panel of cell line and/or PDX models for each tumor type (40). In our case, we demonstrated that fractionated dosing did not Authors' Contributions impact antitumor activity of two targeted PBD ADCs across Conception and design: B. Zheng, X.Q. Yu, H. Zhong, J. Harper, M. Rebelatto, multiple tumor types. Therefore, we believe that these findings P.W. Howard, R. Dixit Development of methodology: B. Zheng, J. Harper, M. Rebelatto, P.W. Howard should be further explored in other tumor models and ultimately Acquisition of data (provided animals, acquired and managed patients, in patients. provided facilities, etc.): M.J.M. Hinrichs, S. Breen, C. Chen Similar to efficacy models, toxicology studies in healthy ani- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mals are not always predictive of the dose-limiting toxicities in computational analysis): P.M. Ryan, B. Zheng, X.Q. Yu, M. Gunsior, H. Zhong, patients (41). Although there are many variables that impact the J. Harper, K. Vashisht, M. Rebelatto, C. Chen clinical predictive value of toxicology studies, one of the major Writing, review, and/or revision of the manuscript: M.J.M. Hinrichs, P.M. Ryan, S. Afif-Rider, X.Q. Yu, M. Gunsior, H. Zhong, K. Vashisht, determinants is the organ system involved. In the case of cytotoxic M. Rebelatto, P.C. Ryan, C. Chen, P.W. Howard, R. Dixit agents—where bone marrow suppression is a common toxicity Administrative, technical, or material support (i.e., reporting or organizing clearly related to the mechanism of action (42)—animal models data, constructing databases): M.J.M. Hinrichs, P.M. Ryan, B. Bezabeh, are generally predictive of hematologic toxicity in patients (43). M. Reed, C. Chen Therefore, it seems probable that the benefits of fractionated Study supervision: S. Afif-Rider, H. Zhong, M. Reed, R. Dixit fi dosing on bone marrow suppression observed in our study could Other (manufacture, puri cation and analysis of pyrrolobenzodiazepine- fi containing antibody drug conjugates for this study): N. Patel translate to patients. If so, this nding could be important to Other (synthesis of materials): L. Masterson clinical trial design of PBD-based ADCs, where bone marrow Other (SG3249 synthesis): A. Tiberghien suppression has been reported as a significant dose-limiting Other (design of SG3400 and supply of material for study): P.W. Howard toxicity humans (14, 36, 44). However, toxicology studies are Other (provided antibodies, antibody drug conjugates and related support- generally less reliable predictors of kidney toxicity, with reports ing information): N. Dimasi that monkeys, in particular, grossly over predict the incidence of renal injury in humans (43). Therefore, it is possible that the Acknowledgments benefits of fractionated dosing on kidney injury might not trans- We thank Moucun Yuan, Elizabeth Dompkowski, Michael Waldron, late to patients—especially given that renal toxicity has not been a and William Mylott Jr of PPD Laboratories (Richmond, VA) for carrying major adverse effect of PBD ADCs in the clinic (14, 44). Despite out LC-MS/MS studies. the difficulties associated with use of preclinical models to under- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked stand exposure-response relationships, it is still much easier than advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate trying to identify the pharmacokinetic drivers of safety and this fact. efficacy in patients—where clinical pharmacokinetic data are largely derived from a single-dose schedule (45) and most Received January 25, 2017; revised April 5, 2017; accepted June 13, 2017; patients have concurrent medical conditions and/or medications published OnlineFirst June 13, 2017.

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Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine-Containing Antibody Drug Conjugates

Mary Jane Masson Hinrichs, Pauline M. Ryan, Bo Zheng, et al.

Clin Cancer Res Published OnlineFirst June 19, 2017.

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